In previous studies we showed that Mitf encodes a bHLH-Zip transcription factor that is required for melanocyte, osteoclast, and mast cell function. Subsequently, human MITF was cloned and shown to be mutated in a common human pigmentation and hearing disorder, Waardenburg syndrome type 2 (WS2). The cloning of mouse Mitf thus led to the identification of a new important human disease gene. We made germ line mutations in three genes that are closely related to Mitf and whose protein products interact with MITF (Tfe3, Tfeb, and Tfec). We also generated mice that carried different combinations of these four mutations. Our studies showed that Tfeb is required for placental vascularization, while Tfe3 functions in the osteoclasts. Surprisingly, we failed to show any genetic interaction between these four genes. This was unexpected based on the MYC-MAX-MAD paradigm. Both Tfeb and Tfe3 are human disease genes and our studies may help to show how these proteins function in human disease. We also showed that Mitf expression is complex; there are at least 13 different isoforms. Others have shown that MITF functions in a number of signal transduction pathways and undergoes several posttranslational modifications. To further evaluate the requirement for these modifications in vivo, we are using BAC recombineering to create mutations within the mouse Mitf gene that block these modifications and then analyze their effects in animals carrying the BAC and a null allele of Mitf. We are also continuing a sensitized, F1 dominant screen for suppressors and enhancers of MITF. In preliminary studies, we have identified a very interesting intragenic suppressor that we are now characterizing. itch represents one of the few single gene mouse models of human autoimmune disease. Previously, we showed that itch encodes a novel HECT-domain-containing E3 ligase. In recent collaborative studies, we showed that loss of itch function leads to a bias towards Th2 differentiation of T cells in vitro. In recent transplantation studies we also showed that cells residing in the bone marrow of itch mice can confer the disease to lethally irradiated wild type hosts. We are now determining whether a particular class of lymphocytes is sufficient for itch disease and whether itch mice have defects in macrophage function that might predispose them to the Th2 bias. itch is the mouse ortholog of Drosophila Suppressor of deltex, Su(dx), a negative regulator of Notch signaling. In recent studies, we found that mice carrying an activated Notch1 transgene have an autoimmune disease that is similar to that seen in itch mice. We also showed that itch mice carrying the activated Notch1 transgene have an autoimmune disease that is more severe and occurs much earlier than itch or transgenic mice alone. These findings are very interesting because they suggest that both itch and Notch1 may function in the same autoimmune disease pathway. Further characterization of this pathway may have important implications for treating human autoimmune diseases. Finally, we are also determining the functional overlap between ITCH and the closely related E3 ligases WWP1 and WWP2 by examining the phenotype of mice carrying combinations of mutations in these three genes. Mice homozygous for the semidominant Crc mutation die during development from severe neural tube defects similar to those observed in Lp mice. Crc and Lp genetically interact at the level of neural tube closure, suggesting that they function in the same signaling pathway. Lp encodes a homolog of the Drosophila vang/stbm gene (Vangl2), a potential member of the Wnt signaling pathway, while Crc encodes Scrb1, a PDZ domain-containing gene that is the ortholog of Drosophila scribble. In flies, scribble is required for the correct localization of apical domain proteins and for the generation of epithelial polarity.